PROJECT SUMMARY Heart failure (HF) is a growing concern among researchers, with rates expected to increase by 25% by 2030. HF is an incredibly complex disease with many pathological features including cardiomyocyte hypertrophy, contractile dysfunction and fibrotic remodeling. HF has complex etiologies, with common risk factors such as hypertension and diabetes being in and of themselves multi-factorial. Consequently, there is a tremendous amount of heterogeneity in human populations in both disease onset and progression which mask the demonstrated strong genetic component of common forms of HF. As a result of this heterogeneity, human genome-wide association studies have only been able to recover a handful of significant loci. Recently, the PI's group described a population of mice in which over 30 loci for HF-related phenotypes were identified and which demonstrated significant overlap (50%) with significant or suggestive HF-associated loci in humans. This proposal outlines an extension of this panel of mice to explore the epigenome of the heart both before and after catecholamine stimulation. Using novel experimental techniques and computational tools, the proposal seeks to identify important genes and pathways which control DNA methylation. It also seeks to connect DNA methylation to changes in HF-related phenotypes as well as outlining an extensive career development plan for Dr. Rau to complete his training under the mentorship of Dr. Wang and transition to an independent academic position by establishing a multi-disciplinary research program in cardiovascular genetics and genomics. During the K99 phase of this award, Dr. Rau will analyze the methylomes of 88 strains of mice both before and after catecholamine stimulation using reduced representation bisulfite sequencing. Research will focus on the use of methylation and phenotypic data to identify CpG-phenotype associations at epigenome-wide association study loci. Genotype-methylation associations will be examined to identify loci within which DNA mutations drive large differences in DNA methylation across the genome. By combining these loci with the significant amount of data previously gathered in this panel, Dr. Rau will predict causal genes and pathways implicated in HF. Based on preliminary results, during the K99 portion of the award the PI will also perform in vivo functional studies using the CRISPR/Cas9 gene knockout system of two high-confidence candidate genes: Mospd3, which regulates heart weight and Serpina3n, which is implicated in the regulation of over 1800 CpGs. During the R00 portion, the PI will combine the genes identified during the K99 portion with his training in in vivo validation to identify novel genes and pathways which contribute to heart failure. The overall goal of the proposed studies is to integrate systems biology, epigenetics and molecular analyses to lead to deeper understandings of the genetic pathways which regulate DNA methylation and HF- related phenotypes.